Mammalian cells possess two major intrinsic mechanisms, apoptosis and senescence, to protect against uncontrolled proliferation when oncogenic mutations occur. To define whether telomerase-induced breaching of the senescence barrier would enable oncogenic Ras to promote uncontrolled cell cycle progression in cells that commonly (bronchial epithelial cells, BECs) or rarely (mammary epithelial cells, MECs) harbor oncogenic ras, we introduced oncogenic H-rasV12 into hTERT immortalized BECs and MECs. To our surprise, rather than promoting cell cycle transit, increased expression of Ras led to G1 cell cycle arrest and an autophagy-like phenotype in both cell types - a response that was independent of the canonical autophagy promoter beclin 1. During extended observation in culture (56 days), a subpopulation of H-RasV12 expressing cells reversed the autophagic phenotype, escaped from cell cycle blockade and formed multilayered foci. The data are in accord with a scenario in which activated Ras triggers at least two contradictory signals: one leading to oncogenesis and the second promoting non- canonical autophagy, cell cycle arrest and cell death. Here we propose 3 specific aims to test the hypothesis that that Ras-induced autophagic arrest and cell degradation represents a previously cryptic third line of defense against Ras oncogenesis that became apparent in cells that had breached the senescence barrier. We further hypothesize that the cell-lineage-dependent status of key cancer-related pathways determines whether Ras-arrested autophagic cells die - or overcome proliferative blockade and undergo malignant transformation. Aim1. Determine whether Ras-induced autophagic reactions are required for Ras- dependent cell cycle arrest by modulating key regulators of autophagy. Aim 2. Identify pathways downstream of activated Ras leading to autophagic cell cycle arrest by systematically inhibiting steps in the PI3K/Akt/mTOR and BRAF/MAPK/ERK pathways. Aim 3. Determine whether activation of c-Myc or eIF4F, suppression of p53 or p16Ink4 - or combinations of these oncogenic interventions - contribute to the cell-context dependent mechanism of escape from oncogenic Ras-mediated autophagy and cell cycle arrest. PUBLIC HEALTH RELEVANCE: Our work tests the idea that cells can activate a second line of defense, autopaghy, - besides senescence and apoptosis- against Ras oncogenesis, and pursues identifying the mechanism by which cancer escapes this checkpoint. This work provides the opportunity to study negative regulators of autophagy as a novel avenue of new targets for therapeutics or chemo-preventive agents to improve human health.